EP2590659B1 - Gelsolin-enriched blood for therapeutic use - Google Patents
Gelsolin-enriched blood for therapeutic use Download PDFInfo
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- EP2590659B1 EP2590659B1 EP11764081.3A EP11764081A EP2590659B1 EP 2590659 B1 EP2590659 B1 EP 2590659B1 EP 11764081 A EP11764081 A EP 11764081A EP 2590659 B1 EP2590659 B1 EP 2590659B1
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- blood
- gold particles
- gelsolin
- protein
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Images
Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/52—Cytokines; Lymphokines; Interferons
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5406—IL-4
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/5428—IL-10
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5437—IL-13
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/545—IL-1
Definitions
- Degenerative joint diseases are of great importance in both humans and animals.
- arthrosis occurs idiopathically (with known risk factors) in the elderly patient, or may be caused by post-traumatic conditions in younger patients as a complication.
- both forms have the same clinical symptoms, including joint pain and impaired function, and often result in a severely compromised quality of life for the affected patient.
- Various causes such as overloading, incorrect loading, joint instability or even infections lead to a mechanical and enzymatic damage to the articular cartilage with apoptosis of the chondrocytes, as well as loss of collagen type II and proteoglycans.
- cartilage degeneration is central to the pathogenesis, the disease affects not only the cartilage, but also the joint capsule and the subchondral bone.
- the breakdown products enter the synovium and lead to synovitis.
- damage to the joint capsule can directly lead to a release of inflammatory mediators and a severely traumatized joint capsule also results in a joint instability.
- the subchondral bone plate adapts to increased bone density.
- this sclerosis makes the bone stiffer and more brittle. On the one hand, this leads to a decrease in the ability to absorb shock, which puts more stress on the overlying cartilage and, on the other hand, shearing forces at the subchondral bone plate junction and mineralized cartilage.
- proinflammatory cytokine tumor necrosis factor alpha
- IL-1 interleukin 1
- IL-6 interleukin 6
- PGE2 prostaglandin E2
- the proinflammatory (inflammatory) cytokines TNF ⁇ , IL-1 and IL-6 are secreted by synovial membrane B synoviocytes (synovial membrane, inflammatory cells in synovial membrane and chondrocytes) and stimulate the release of matrix metalloproteinases (MMPs) and aggrecanases as well as other inflammatory mediators such as prostaglandins (PGE2) or nitrite oxide (NO).
- MMPs matrix metalloproteinases
- PGE2 prostaglandins
- NO nitrite oxide
- Metalloproteases are enzymes that degrade the matrix of cartilage (type II collagen, proteoglycans, etc.). II-1 and TNF ⁇ also directly inhibit the production of type II collagen.
- cytokine inhibitors or chondroprotective drugs are the treatments with cytokine inhibitors or chondroprotective drugs called "disease-modifying drugs" (Qvist et al., 2008). These include therapies in which the body's own (autologous) proteins are extracted from the patient's blood and re-administered to this patient as an individual medication.
- the internal structures of the syringe including particles arranged in the syringe, in particular glass beads impregnated with chromium sulfate, are coated with immobilized inducers intended to stimulate the biosynthesis of the desired proteins.
- inducers intended to stimulate the biosynthesis of the desired proteins.
- immunoglobulins in particular immunoglobulin G, are provided as such inducers in order to stimulate the monocytes contained in the blood to form anti-inflammatory proteins.
- the syringe is filled with a body fluid of a patient and incubated.
- the therapeutically active protein is formed in the body fluid.
- the thus enriched body fluid can be stored sterile in the syringe and, if necessary, the patient directly without further treatment or, for example, after centrifugation and / or sterile filtration can be recycled.
- One aspect of the present disclosure is to further develop such a method such that the proteins produced are present in significantly higher amounts and that undesirable side effects are avoided.
- the disclosure shows the provision of a method of the type mentioned, in which the container contains gold particles.
- 'container' is hereafter a closable container or a closable container for storing liquids for a certain time, wherein the container or container is close to the liquid to be absorbed.
- the combination of gold particles with a human or animal autologous or homologous body fluid, in particular a self-blood sample, used in a closed system for the first time in a closed system surprisingly not only provides (i) a significantly higher concentration of the desired proteins in comparison to the previously known method Case of blood in particular of the cytokines (especially IL-1, IL-6, IL-8, IL-10, G-CSF, MCP-1, MIP-1, RANTES, TNF-alpha, GRO-alpha, MCP-3 (Ii) also a better quality of the proteins concerned due to an inhibition of physiological protein aging during the incubation period and (iii) also a significant accumulation of the protein gelsolin.
- Gelsolin is an ubiquitous in all animal (including human) cells and also extracellular (eg in the blood plasma) actin-binding protein that fragmented Ca 2+ -dependent actin filaments and prevents repolymerization, and that has a key role in the regulation of actin filament structure and - Dismantling processes has / met.
- Gelsolin has been discovered and identified in cytoplasmic extracts, and its ubiquitous presence and phylogenetic conservation in motile cells speaks for its essential role as an intracellular regulatory protein. Only later was it discovered that gelsolin also occurs in mammalian blood plasma and depolymerizes actin there.
- Plasma gelsolin is an isoform of cytoplasmic gelsolin (cGelsolin, cGS) and differs structurally in that it has an additional 23 amino acids at the N-terminal end.
- the physiological function of plasma gelolin is still the subject of much research (DiNubriu, 2007 and literature cited therein).
- Gelsolin regulates important cellular functions such as cell motility, phagocytosis, apoptosis and activation of platelets (Silacci et al., 2004, Trickey et al., 2004). In people with rheumatoid arthritis, the plasma concentration of gelsolin is reduced (Osborn et al., 2008).
- the proteins produced by the method according to the present invention may (but need not) be administered directly to the patient, ie without further manipulation - such as the other components of the liquid in the container. Centrifugation, sterile filtration or transfer to other containers - to be re-applied. This avoids contamination of the protein solution and minimizes the risk of patient infection when administering the proteins.
- the gold particles are removed from the body fluid, for example the serum, and discarded.
- the body fluid for example the serum
- both the gold particles and body cells (for example blood cells) and other insoluble aggregates are removed from the body fluid (for example the blood) after in vitro incubation and discarded.
- body fluid for example the blood
- Such treated autologous human body fluids and especially serums are excellently tolerated and side effects are not expected.
- the gold particles used in the process preferably have a defined structure and / or a defined size. Micro- and / or nanoparticles with a particle size between 10 nanometers to 500 micrometers are particularly suitable.
- the gold particles are to be removed and discarded after the in vitro incubation from the body fluid, for example the serum, preferably gold particles with the size of about 1 .mu.m, more preferably in one, are used in the method Amount of 10 3 - 10 4 gold particles per 10 ml.
- containers for example a syringe
- a capacity of about 10 ml have proven useful for this application.
- the gold particles are present in the container in an amount of 0.3 mg per 1 ml of body fluid. Concentrations of 0.1 to 10 mg per 1 ml are basically suitable and intended.
- the internal structure of the container is preferably free of anticoagulants such as heparin, citrate, EDTA or CPDA, because it has surprisingly been found in the context of the work on which this invention is based that fewer proteins are biosynthesized in the presence of such substances than in the absence.
- anticoagulants such as heparin, citrate, EDTA or CPDA
- a container is also a closable bag into consideration, because it can be handled and stored more flexible especially for larger volumes than a syringe with a comparable volume, and because it can be coupled technically simple and safe with a syringe, so that the filling and emptying can be done over this.
- the process according to the invention is particularly suitable for the production (biosynthesis) and accumulation of proteins from blood cells. Therefore, in particular blood is provided as body fluid.
- the therapeutically active protein gelsolin is particularly well, i. produce and accumulate in significant amounts with the method according to the invention. Therefore, the process according to the invention is intended in particular for the recovery of gelsolin.
- a solution of the stated object is also to provide a container for in vitro biosynthesis and enrichment (in vitro induction) of therapeutically active proteins in a body fluid, which is characterized in that the container contains gold particles and for receiving, storage and Re-delivery of a body fluid sample is suitable, and that it can be coupled with a hollow needle (cannula, injection needle) such that its contents can be injected by means of this hollow needle (cannula, injection needle).
- a hollow needle cannula, injection needle
- the previously described protein production process can be carried out and use the advantages associated with this.
- the proteins produced can be brought directly to the desired application site by means of the dockable hollow needle, in particular be introduced into a human or animal body.
- the gold particles preferably have a defined structure and / or a defined size. Micro and / or nanoparticles (particle size between 10 nanometers and 500 micrometers) are particularly suitable.
- An appropriate amount of gold particles in the container is 0.1 mg to 10 mg per 1 ml of body fluid, preferably 0.3 mg per 1 ml of body fluid.
- the container is preferably a syringe or a bag.
- the syringe has the advantage that it can serve not only as an incubation vessel, but at the same time as a sampling device for obtaining the body fluid and / or as an application tool for the administration of the proteins to a patient.
- the bag has the advantage that it can be handled and stored more flexibly with larger volumes than a syringe of comparable volume and can be coupled with a syringe for filling and emptying.
- the gold particles e.g., Gold Microcarriers from BioRad Laboratories, Cat. 165-2264.
- the syringe in particular the inner wall of the syringe barrel and the syringe barrel located in the cylinder, is preferably made of a plastic, for example polystyrene, polyethylene, polyvinyl chloride, polypropylene (neutral S-Monovetten, Sarstedt) or a similar substance or mixtures thereof.
- a plastic for example polystyrene, polyethylene, polyvinyl chloride, polypropylene (neutral S-Monovetten, Sarstedt) or a similar substance or mixtures thereof.
- the container is particularly well suited for the production (biosynthesis) and enrichment of proteins, in particular of gelsolin, from blood cells. Therefore, in particular blood is provided as body fluid.
- the proteins prepared by the method according to the invention in a body fluid can be used together with the body fluid and the gold particles or without the gold particles as a medicament for the treatment of diseases.
- the protein-enriched and, in particular, cytokinin-enriched and gelsolin-enriched body fluids, in particular blood sera, which are produced using the methods according to the invention, represent a safe, cost-effective and rapidly produced alternative with low side effects compared to comparable conventional pharmaceutical preparations.
- the present invention relates to a cytokinin and gelatin-enriched blood for use as a medicament or for the preparation of a medicament obtainable by taking blood into a container containing gold particles, preferably gold particles of about 1 ⁇ m in size, and preferably in a quantity of 10 3 - 10 4 gold particles per 10 ml container (or 0.3 mg of gold particles with a diameter of 1 ⁇ m per 1 ml of blood / container) that this mixture of blood, and gold particles is incubated (for example over 12-72 hours, preferably over 24 hours and at 20 ° C to 41 ° C, preferably at 37 ° C) and then that the gold particles and preferably (ie optionally) also blood cells and other insoluble constituents from the body fluid, especially the blood, are removed and discarded ( preferably by centrifugation and / or sterile filtration).
- gold particles preferably gold particles of about 1 ⁇ m in size, and preferably in a quantity of 10 3 - 10 4 gold particles per 10 ml container (or 0.3
- the disclosure also relates to the use of autologous or homologous blood and gold particles in combination as a pharmaceutical or for the manufacture of a medicament.
- subject of the present invention is also a mixture of autologous or homologous blood and gold particles for use as drugs, or a drug comprising the mixture autologous or homologous blood and gold particles including the proteins enriched therein as a combination of active ingredients.
- the disclosed pharmaceuticals enable a particularly simple, inexpensive, low-risk, and effective treatment.
- the medicaments described are particularly suitable for the treatment of degenerative tissue diseases, especially of arthroses and tendinoses.
- the drug-incubated and subsequently particle-depleted drug is well-suited and contemplated for the treatment of osteoarthritis and other diseases associated with tissue degeneration and / or associated with gelsolin deficiency.
- a particular embodiment of the medicaments according to the invention is characterized in that the blood incubated with gold particles after incubation has a content of gelsolin which is at least twice the corresponding standard blood level for gelsolin.
- corresponding standard blood level for gelsolin in the present context stands for the human medical or veterinary standard value for gelsolin in the blood of the group of patients to be treated with the drug.
- the patient group is characterized by its zoological species and race, sex and age.
- This Gelsolin-rich medicine is intended for the treatment of osteoarthritis associated with a lack of gelsolin in the patient's blood.
- the incubated blood-gold mixture can be administered in whole or in part. If necessary, it may be subjected to centrifugation and / or sterile filtration prior to administration to the (animal or human) patient, e.g. To remove cells and cell fragments from the blood and at the same time to reduce the injection volume.
- Example 1 Verification of Protein Production in a Blood Pool according to the disclosed method and in a disclosed container by means of MID-FTIR spectroscopy method and multiplex analysis
- the measuring principle of Fourier transform infrared spectroscopy is based on the irradiation of a substance with electromagnetic waves, whereby certain frequency ranges are absorbed. Since infrared radiation is energetically in the range of vibrational levels of molecular bonds, absorption leads to vibrational excitation of the bonds. This is visible in the form of deflections in the measured spectrum (diagram). Since the energies or frequencies necessary for this are characteristic of the respective bonds, materials can also be identified and structures can be elucidated.
- the FTIR spectroscopy is particularly suitable for the analysis of structural, reaction-induced changes in a biological macromolecule. It can be used to study biological systems, in particular protein-containing aqueous liquids. The sample is neither altered nor destroyed and it can be used under native conditions of the biomolecule,. i.e. It is possible to measure the "actual state" because neither a fixation nor a different preparation of the samples is necessary.
- the MID-FTIR spectroscopy method allows automated and reproducible detection and quantification of changes in protein conformation and determination of protein concentration. It is already possible to detect very low protein concentrations (below 0.1 mg / ml) and lowest conformational changes.
- the blood samples were analyzed with and without gold particles in the "as is" state in a highly accurate biocompatible and suitable for aqueous samples spectroscopic measuring cell (transmission cell).
- concentration of the dissolved protein and its secondary structure were automatically determined from each measured sample.
- Fig. 1 shown graphically. They show that the samples incubated according to the invention in the presence of gold particles behave biologically differently than the control samples.
- the measured values of all samples at time T0 are marked in blue color and are predominantly in the upper left quadrant.
- the measured values after carrying out the method according to the invention with an incubation period of 24 hours are marked in green color and are predominantly in or near the left lower quadrant. This indicates that physiological protein aging is inhibited by the addition of gold during blood incubation.
- the measured values of the control samples after an incubation period of 24 hours are marked in red and are predominantly in the upper right quadrant. This shift to the right indicates an aging of the protein structure.
- invention had a significant increase in the gelsolin concentration (factor 10) after 24 hours, while the control samples (“ control T24 “ and " StdT-T24 ”) did not show gelatinization but rather gelsonline depletion was determined.
- concentrations of tumor necrosis factor alpha (TNF- ⁇ ), macrophage chemotactic protein (MCP-3) and growth-regulated oncogenic alpha (“GRO ⁇ ”) were also significant in the samples treated according to the invention (“invention") (TNF-a: factor 30 -40, MCP-3: factor 20-30) or at least significantly (GROa: factor 2) higher than in the control samples ("Control-T24” and "StdT-T24”).
- a container in the form of a gold particle-containing syringe was filled with blood of the animal in question and incubated for 24 hours at a temperature in the range of 37 ° C. After the incubation period, the drug in the form of the blood in the syringe with the synthesized and enriched in the meantime proteins, especially cytokines and gelsolin, and the gold particles was completed and could be used directly and immediately.
- the drug was manufactured in a syringe, it could be given by injection to the animal in question without being refilled and thus without the risks of contamination and material loss.
- the drug injections were given to the respective horse one week apart.
- the first administration was at time T24, the second, third and fourth after week 1, after week 2 and after week 3.
- the drug-containing syringes for the second, third and fourth applications were stored at minus 20 ° C until use ,
- the drug injections were given to the respective horse one week apart.
- the first administration was at time T24, the second, third and fourth after week 1, after week 2 and after week 3.
- the drug-containing syringes for the second, third and fourth applications were stored at minus 20 ° C until use ,
- Example 3 Preparation of a container according to the invention with gold particles
- the gold particles used were gold powder (particle size 1 ⁇ m, Bio-Rad Laboratories, Kunststoff).
- the gold particles were first sterilized as recommended by the manufacturer: The required amount of gold particles / gold powder, for example 30 mg, was mixed with 1 ml of 70% ethanol, incubated for 10 minutes with gentle stirring (mixer, vortexer), settling after 1 minute centrifuged briefly and then the supernatant removed. Thereafter, the gold particles were redistilled three times with 1 ml of sterile water. washed. After the final wash with final centrifugation and spillage of the supernatant, the gold particles were resuspended in sterile PBS and adjusted to the desired concentration, eg, 60 mg / ml.
- the cartilage-bone samples from the respective femoral heads were removed from the respective animals under aseptic conditions and cut to 8x8x10 mm (length / width / height) blocks.
- the cartilage was macroscopically intact in all explants.
- cartilage-bone sample blocks were placed in DMEM medium supplemented with 10% human serum (HS), 100 U / ml pencillin, 100 ⁇ g / ml gentamycin, and 1.25 U / ml amphotericin B stored.
- HS human serum
- 100 U / ml pencillin 100 ⁇ g / ml gentamycin
- 1.25 U / ml amphotericin B stored.
- the impact load test (impaction test) was carried out on the day of the sampling.
- a cartilage-bone sample block / explant was arranged in a cylindrical drop tower made of polymethyl methacrylate with the dimensions 33 cm height and 4 cm axially with distance below an impact piston under sterile conditions.
- the butt piston had the shape of a cylinder measuring 5 cm high and 3.94 cm in diameter and weighing 493 g.
- the butt piston was threadedly connected to the actual impact disk ("impactor piece"), which had a weight of 7 g, a height / thickness of 1 cm and a diameter of 0.6 cm.
- the impact loading / impacting took place once per cartilage-bone sample block in the "drop tower" by the free fall of the impact piston with impact disk from 15 cm height below the sample.
- the impact load was 0.736 J on a cartilage surface of 28.3 mm 2 .
- the treated bone-cartilage preparations as well as the 0 controls were washed 3 times with PBS and in 12-well culture plates transferred. Each explant was prepared with 3 ml of the respective treatment medium and incubated under standardized conditions (37 ° C, 5% CO 2 ,) in the incubator. The culture medium was changed every 72 hours.
- the protein-raised blood serum prepared according to the present invention was added to the explant of the subject explant group at day 0 and day 7.
- the proteoglycan content in the culture medium was measured in all assays.
- the Blyscan glycosaminoglycan assay from Biocolor Ltd. (Carrickfergus, UK) was used for this purpose.
- the results are in the graph of Fig. 7 shown.
- the proteoglycan content is given as mean glycosaminoglycan (GAG) concentration in ⁇ g / ml medium.
- proteoglycan release shows that in all three groups, namely the 0 controls and the two shock-treated / impacted explant / cartilage-bone preparations, the amount of proteogycan increases with time.
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Description
Offenbart wird ein Verfahren zur Herstellung mindestens eines therapeutisch wirksamen Proteins oder eines Proteingemisches in einem Behältnis, wobei das Behältnis mit einer Körperflüssigkeit gefüllt, inkubiert und das therapeutisch wirksame Protein in der Körperflüssigkeit gebildet wird. Offenbart wird außerdem ein Behältnis für die Durchführung des Verfahrens und Arzneimittel, die die derart hergestellten Proteine als Wirkstoff enthalten.Disclosed is a method for producing at least one therapeutically active protein or a protein mixture in a container, wherein the container filled with a body fluid, incubated and the therapeutically active protein is formed in the body fluid. Also disclosed is a container for carrying out the method and medicaments containing the proteins thus produced as active ingredient.
Degenerative Gelenkerkrankungen haben sowohl beim Menschen als auch bei Tieren eine große Bedeutung. Beim Menschen tritt die Arthrose idiopathisch (mit bekannten Risikofaktoren) beim älteren Patienten auf, oder kann bei jüngeren Patienten als Komplikation posttraumatisch bedingt entstehen. Beide Formen haben aber die gleichen klinischen Symptome, u.a. Gelenkschmerzen und eingeschränkte Funktion, und führen häufig zu einer stark eingeschränkten Lebensqualität des betroffenen Patienten. Verschiedene Ursachen wie Überbelastung, Fehlbelastung, Gelenkinstabilität oder auch Infektionen führen zu einer mechanischen und enzymatischen Schädigung des Gelenkknorpels mit Apoptose der Chondrozyten, sowie Verlust an Kollagen Typ II und Proteoglykanen. Dabei besteht eine Imbalance zwischen Degeneration und Reparation. Obwohl im Mittelpunkt der Pathogenese die Knorpeldegeneration steht, betrifft die Krankheit nicht nur den Knorpel, sondern auch die Gelenkskapsel und den subchondralen Knochen. Bei Knorpelschäden gelangen die Abbauprodukte in die Synovia und führen zu einer Synovitis. Zudem können auch Schäden an der Gelenkskapsel direkt zu einer Freisetzung von Entzündungsmediatoren führen und eine stark traumatisierte Gelenkskapsel resultiert zudem in einer Gelenksinstabilität. Bei hoher und zyklischer Belastung adaptiert sich die subchondrale Knochenplatte mit einer erhöhten Knochendichte. Durch diese Sklerosierung wird aber der Knochen steifer und spröder. Dies führt einerseits zu einer Abnahme der Fähigkeit zur Schockabsorption, was den darüber liegenden Knorpel mehr belastet, und andererseits zu Scherkräften am Übergang subchondrale Knochenplatte und mineralisiertem Knorpel. Bei Pferden mit Arthrose und/oder Osteoarthritis und/oder anderen Gelenkserkrankungen wurden erhöhte Konzentrationen der proinflammatorischen (entzündungsfördernden) Zytokine Tumor Nekrose Faktor alpha (TNFa), Interleukin 1 (IL-1) und Interleukin 6 (IL-6) sowie an Prostaglandin E2 (PGE2) und Metalloproteasen in der Synovia gemessen. Auch bei Menschen mit Arthrose ist die Konzentration der proinflammatorischen Zytokine im Blut und in der Synovia erhöht.Degenerative joint diseases are of great importance in both humans and animals. In humans, arthrosis occurs idiopathically (with known risk factors) in the elderly patient, or may be caused by post-traumatic conditions in younger patients as a complication. However, both forms have the same clinical symptoms, including joint pain and impaired function, and often result in a severely compromised quality of life for the affected patient. Various causes such as overloading, incorrect loading, joint instability or even infections lead to a mechanical and enzymatic damage to the articular cartilage with apoptosis of the chondrocytes, as well as loss of collagen type II and proteoglycans. There is an imbalance between degeneration and reparation. Although cartilage degeneration is central to the pathogenesis, the disease affects not only the cartilage, but also the joint capsule and the subchondral bone. In the case of cartilage damage, the breakdown products enter the synovium and lead to synovitis. In addition, damage to the joint capsule can directly lead to a release of inflammatory mediators and a severely traumatized joint capsule also results in a joint instability. At high and cyclic loading, the subchondral bone plate adapts to increased bone density. However, this sclerosis makes the bone stiffer and more brittle. On the one hand, this leads to a decrease in the ability to absorb shock, which puts more stress on the overlying cartilage and, on the other hand, shearing forces at the subchondral bone plate junction and mineralized cartilage. In horses with osteoarthritis and / or osteoarthritis and / or other joint disorders have been elevated levels of proinflammatory (proinflammatory) cytokine tumor necrosis factor alpha (TNFa), interleukin 1 (IL-1) and interleukin 6 (IL-6) as well as prostaglandin E2 (PGE2) and metalloproteases measured in the synovium. Also, in people with osteoarthritis, the concentration of proinflammatory cytokines in the blood and in the synovium is increased.
Die proinflammatorischen (entzündungsfördernden) Zytokine TNFα, IL-1 und IL-6 werden von den B-Synoviozyten (Synoviocyti secretorii) der Synovialmembran, von Entzündungszellen in der Synovialmembra und von Chondrozyten sezerniert und stimulieren die Freisetzung von Matrixmetalloproteasen (MMP's) und Aggrecanasen sowie von weiteren Entzündungsmediatoren wie Prostaglandinen (PGE2) oder Nitritoxid (NO). Metalloproteasen sind Enzyme, welche die Matrix des Knorpel (Kollagen Typ II, Proteoglykane u.a.) degradieren. II-1 und TNFα hemmen auch direkt die Produktion von Typ II Kollagen.The proinflammatory (inflammatory) cytokines TNFα, IL-1 and IL-6 are secreted by synovial membrane B synoviocytes (synovial membrane, inflammatory cells in synovial membrane and chondrocytes) and stimulate the release of matrix metalloproteinases (MMPs) and aggrecanases as well as other inflammatory mediators such as prostaglandins (PGE2) or nitrite oxide (NO). Metalloproteases are enzymes that degrade the matrix of cartilage (type II collagen, proteoglycans, etc.). II-1 and TNFα also directly inhibit the production of type II collagen.
Die herkömmliche Therapie der degenerativen Gelenkerkrankungen besteht in den meisten Fällen in einer symptomatischen Behandlung der Entzündung, d.h. in einer entweder systemischen oder intraartikulären Hemmung der Entzündung mittels entsprechender Medikamente. Hierzu gehören die am häufigsten eingesetzten intraartikulär applizierten Kortikosteroiden, teilweise in Kombination mit Hyaluronsäure. Zusätzlich werden gelegentlich außerdem Chondroprotektiva verabreicht.Conventional therapy of degenerative joint disease in most cases involves symptomatic treatment of inflammation, i. in either systemic or intra-articular inhibition of inflammation by appropriate drugs. These include the most commonly used intra-articular corticosteroids, sometimes in combination with hyaluronic acid. In addition, occasionally chondroprotective drugs are also administered.
Diese medikamentösen Therapien haben jedoch zahlreiche Nebenwirkungen.However, these drug therapies have numerous side effects.
Eine Alternative zu den symptomatischen Therapien (auf der Basis nicht steroidaler und steroidaler Entzündungshemmer) bilden die Behandlungen mit Zytokin-Inhibitoren oder Chondroprotektiva, die als "disease modifying drugs" bezeichnet werden (Qvist et al. 2008). Hierzu zählen auch Therapien, bei denen körpereigene (autologe) Proteine aus dem Blut des Patienten gewonnen und als individuelles Medikament diesem Patienten wieder verabreicht werden.An alternative to the symptomatic therapies (based on non-steroidal and steroidal anti-inflammatory drugs) are the treatments with cytokine inhibitors or chondroprotective drugs called "disease-modifying drugs" (Qvist et al., 2008). These include therapies in which the body's own (autologous) proteins are extracted from the patient's blood and re-administered to this patient as an individual medication.
Aus der
Die inneren Strukturen der Spritze, wozu auch in der Spritze angeordnete Partikel, insbesondere mit Chromsulfat imprägnierten Glaskügelchen, zählen, sind mit immobilisierten Induktoren beschichtet, die die Biosynthese der gewünschten Proteine anregen sollen. Im Fall von Blut als Körperflüssigkeitsprobe sind als solche Induktoren Immunglobuline, insbesondere Immunglobulin G vorgesehen, um die im Blut enthaltenen Monocyten zur Bildung anti-inflammatorischer Proteine anzuregen.The internal structures of the syringe, including particles arranged in the syringe, in particular glass beads impregnated with chromium sulfate, are coated with immobilized inducers intended to stimulate the biosynthesis of the desired proteins. In the case of blood as a body fluid sample, immunoglobulins, in particular immunoglobulin G, are provided as such inducers in order to stimulate the monocytes contained in the blood to form anti-inflammatory proteins.
Für die Durchführung des Verfahrens wird die Spritze mit einer Körperflüssigkeit eines Patienten gefüllt und inkubiert. Dabei wird das therapeutisch wirksame Protein in der Körperflüssigkeit gebildet.To carry out the method, the syringe is filled with a body fluid of a patient and incubated. In this case, the therapeutically active protein is formed in the body fluid.
Die so angereicherte Körperflüssigkeit kann in der Spritze steril gelagert und bei Bedarf dem Patienten direkt ohne weitere Behandlung oder beispielsweise nach Zentrifugation und/oder Sterilfiltration wieder zugeführt werden. Ein Aspekt der vorliegenden Offenbarung besteht darin, ein solches Verfahren derart weiterzuentwickeln bzw. abzuwandeln, dass die erzeugten Proteine in signifikant höheren Mengen vorliegen, und dass unerwünschte Begleiterscheinungen vermieden sind. Die Offenbarung zeigt die Bereitstellung eines Verfahrens der eingangs genannten Art, bei dem das Behältnis Goldpartikel enthält.The thus enriched body fluid can be stored sterile in the syringe and, if necessary, the patient directly without further treatment or, for example, after centrifugation and / or sterile filtration can be recycled. One aspect of the present disclosure is to further develop such a method such that the proteins produced are present in significantly higher amounts and that undesirable side effects are avoided. The disclosure shows the provision of a method of the type mentioned, in which the container contains gold particles.
Der Begriff 'Behältnis' steht im Folgenden für ein verschließbares Gefäß oder einen verschließbaren Behälter zur Aufbewahrung von Flüssigkeiten für eine bestimmte Zeit, wobei Gefäß bzw. Behälter dicht gegenüber der aufzunehmenden Flüssigkeit ist.The term 'container' is hereafter a closable container or a closable container for storing liquids for a certain time, wherein the container or container is close to the liquid to be absorbed.
Der Einsatz von Gold als Arzneimittel ist in der Medizin schon lange bekannt. Ende des 19. Jahrhunderts wurde Gold vor allem als Arzneimittel zur Behandlung von Tuberkulose eingesetzt. Aufgrund der falschen Annahme, dass die rheumatoide Arthritis ebenfalls eine Infektionserkrankung sei, wendete man Gold als Arzneimittel auch bei diesem Leiden an. Die Therapie war erfolgreich und wurde dann über viele Jahre bis in die heutige Zeit angewendet (Kean and Kean 2008). Eine in vitro Studie an humanen Chondrozyten hat gezeigt, dass Goldverbindungen (Aurothiomalat) die Produktion von Nitrit Oxid (NO) der Chondrozyten hemmt. Nitrit Oxid vermittelt (mediiert) die destruktiven Effekte der proinflammatorischen Zytokine IL-1 und TNFα (Green et al. 2006). Dies führt zu einer verminderten Kollagen- und Proteoglykanproduktion, zur Chondrozytenapoptose und zu einer Stimulation der Metalloproteasen (Vuolteenaho et al. 2005).The use of gold as a medicine has long been known in medicine. At the end of the 19th century, gold was used primarily as a drug to treat tuberculosis. Due to the false assumption that rheumatoid arthritis is also an infectious disease, gold was also used as a medicine in this condition. The therapy was successful and was then used for many years to the present day (Kean and Kean 2008). An in vitro study on human chondrocytes has shown that gold compounds (aurothiomalate) inhibit the production of nitrite oxide (NO) from chondrocytes. Nitrite oxide mediates (mediates) the destructive effects of proinflammatory cytokines IL-1 and TNFα (Green et al., 2006). This leads to reduced collagen and proteoglycan production, to chondrocyte apoptosis and to stimulation of metalloproteases (Vuolteenaho et al., 2005).
In den im Stand der Technik bekannten therapeutischen Ansätzen wird das Gold entweder intramuskulär oder per os appliziert. Mit diesen Applikationsmethoden und der entsprechenden Galenik von Gold werden die aus den in vitro-Studien bekannten gewünschten Effekte jedoch nur ansatzweise erreicht.In the therapeutic approaches known in the art, gold is administered either intramuscularly or per os. With these application methods and the corresponding galenics of gold, however, the desired effects known from the in vitro studies are only partially achieved.
Die in dem offenbarungsgemässen Verfahren erstmals eingesetzte Kombination von Goldpartikeln mit einer menschlichen oder tierischen autologen oder homologen Körperflüssigkeit, insbesondere einer Eigenblutprobe, in einem abgeschlossenen System liefert überraschenderweise nicht nur (i) eine im Vergleich zu dem vorbekannten Verfahren deutlich höhere Konzentration der gewünschten Proteine, im Fall von Blut insbesondere der Zytokine (vor allem IL-1, IL-6, IL-8, IL-10, G-CSF, MCP-1, MIP-1, RANTES, TNF-alpha, GRO-alpha, MCP-3, MIF und IL-1RA), sondern (ii) auch eine bessere Qualität der betreffenden Proteine aufgrund einer Hemmung der physiologische Proteinalterung während der Inkubationszeit und (iii) außerdem eine signifikante Anreicherung des Proteins Gelsolin. Gelsolin ist ein ubiquitär in allen tierischen (inklusive menschlichen) Zellen und auch extrazellulär (z.B. im Blutplasma) vorhandenes Aktinbindendes Protein, das Ca2+-abhängig Aktinfilamente fragmentiert und eine Repolymerisation verhindert, und das eine Schlüsselfunktion bei der Regulation der Aktinfilament-Aufbau und -Abbauprozesse hat/erfüllt. Gelsolin wurde in zytoplasmatischen Extrakten entdeckt und identifiziert, und seine ubiquitäre Präsenz und phylogenetische Konservierung in freibeweglichen Zellen spricht für seine essentielle Rolle als ein intrazelluläres regulatorisches Protein. Erst später wurde entdeckt, dass Gelsolin auch im Blutplasma von Säugern vorkommt und dort Aktin depolymerisiert. Dieses sogenannte Plasmagelsolin (pGelsolin, pGS) ist eine Isoform des zytoplasmatischen Gelsolins (cGelsolin, cGS) und unterscheidet sich von diesem strukturell dadurch, dass es am N-terminalen Ende zusätzliche 23 Aminosäuren aufweist. Die physiologische Funktion von PlasmaGelsolin ist nach wie vor Gegenstand zahlreicher Forschungsarbeiten (DiNubriu, 2007 und darin zitierte Literatur). Gelsolin reguliert so wichtige Zellfunktionen wie Zellmotilität, Phagozytose, Apoptose und Aktivierung von Thrombozyten (Silacci et al. 2004, Trickey et al. 2004). Bei Menschen mit rheumatoider Arthritis ist die Plasmakonzentration von Gelsolin vermindert (Osborn et al. 2008). Auch bei anderen Erkrankungen mit Gewebedegenerationen und insbesondere bei Sepsis ist die Plasmakonzentration von Gelsolin vermindert (Suhler et al. 1997, Osborn et al. 2008, Lee et al. 2007). Im Stand der Technik vorhandene Erkenntnisse weisen darauf hin, dass PlasmaGelsolin als Puffer dient, um überschießende Entzündungsreaktionen des Körpers abzufangen (DiNubile 2008).The combination of gold particles with a human or animal autologous or homologous body fluid, in particular a self-blood sample, used in a closed system for the first time in a closed system surprisingly not only provides (i) a significantly higher concentration of the desired proteins in comparison to the previously known method Case of blood in particular of the cytokines (especially IL-1, IL-6, IL-8, IL-10, G-CSF, MCP-1, MIP-1, RANTES, TNF-alpha, GRO-alpha, MCP-3 (Ii) also a better quality of the proteins concerned due to an inhibition of physiological protein aging during the incubation period and (iii) also a significant accumulation of the protein gelsolin. Gelsolin is an ubiquitous in all animal (including human) cells and also extracellular (eg in the blood plasma) actin-binding protein that fragmented Ca 2+ -dependent actin filaments and prevents repolymerization, and that has a key role in the regulation of actin filament structure and - Dismantling processes has / met. Gelsolin has been discovered and identified in cytoplasmic extracts, and its ubiquitous presence and phylogenetic conservation in motile cells speaks for its essential role as an intracellular regulatory protein. Only later was it discovered that gelsolin also occurs in mammalian blood plasma and depolymerizes actin there. This so-called plasma gelsolin (pGelsolin, pGS) is an isoform of cytoplasmic gelsolin (cGelsolin, cGS) and differs structurally in that it has an additional 23 amino acids at the N-terminal end. The physiological function of plasma gelolin is still the subject of much research (DiNubriu, 2007 and literature cited therein). Gelsolin regulates important cellular functions such as cell motility, phagocytosis, apoptosis and activation of platelets (Silacci et al., 2004, Trickey et al., 2004). In people with rheumatoid arthritis, the plasma concentration of gelsolin is reduced (Osborn et al., 2008). The plasma concentration of gelsolin is also reduced in other diseases with tissue degeneration and in particular in sepsis (Suhler et al., 1997, Osborn et al., 2008, Lee et al., 2007). Evidence from the prior art indicates that plasma gel serves as a buffer to counteract excessive inflammatory responses of the body (DiNubile 2008).
Die mit dem offenbarungsgemässen Verfahren hergestellten Proteine, insbesondere Gelsolin und Zytokine, können (müssen jedoch nicht) zusammen mit den anderen Bestandteilen der in dem Behältnis befindlichen Flüssigkeit dem Patienten direkt, das heißt ohne weitere Manipulation - wie z.B. Zentrifugation, Sterilfiltration oder Umfüllen in andere Behälter - wieder appliziert werden. Dadurch wird eine Kontamination der Protein-Lösung vermieden und das Risiko einer Infektion des Patienten bei der Verabreichung der Proteine wird minimiert.The proteins produced by the method according to the present invention, in particular gelsolin and cytokines, may (but need not) be administered directly to the patient, ie without further manipulation - such as the other components of the liquid in the container. Centrifugation, sterile filtration or transfer to other containers - to be re-applied. This avoids contamination of the protein solution and minimizes the risk of patient infection when administering the proteins.
Bei einer Variante des offenbarungsgemässen Verfahrens werden die Goldpartikel nach der in vitro Inkubation aus der Körperflüssigkeit, beispielsweise dem Serum entfernt und verworfen. Das hat den Vorteil, dass goldinduzierte Nebenwirkungen bei oder nach der Verabreichung dieser Körperflüssigkeit, beispielsweise dieses Serums, komplett vermieden werden.In a variant of the method according to the invention, after the in vitro incubation, the gold particles are removed from the body fluid, for example the serum, and discarded. This has the advantage that gold-induced side effects during or after the administration of this body fluid, such as this serum, completely avoided.
Bei einer ebenfalls bevorzugten Variante des offenbarungsgemässen Verfahrens werden sowohl die Goldpartikel als auch Körperzellen (beispielsweise Blutzellen) und andere unlöslichen Aggregate nach der in-vitro Inkubation aus der Körperflüssigkeit (beispielsweise dem Blut) entfernt und verworfen. Derartig aufbereitete autologe humane Körperflüssigkeiten und insbesondere Seren sind hervorragend verträglich, und Nebenwirkungen sind nicht zu erwarten.In a likewise preferred variant of the method according to the invention, both the gold particles and body cells (for example blood cells) and other insoluble aggregates are removed from the body fluid (for example the blood) after in vitro incubation and discarded. Such treated autologous human body fluids and especially serums are excellently tolerated and side effects are not expected.
Die in dem Verfahren eingesetzten Goldpartikel weisen vorzugsweise eine definierte Struktur und/oder eine definierte Größe auf. Mikro- und/oder Nanopartikel mit einer Partikelgröße zwischen 10 Nanometern bis 500 Mikrometern sind besonders geeignet.The gold particles used in the process preferably have a defined structure and / or a defined size. Micro- and / or nanoparticles with a particle size between 10 nanometers to 500 micrometers are particularly suitable.
Bei den Varianten des offenbarungsgemässen Verfahrens, bei denen die Goldpartikel nach der in vitro Inkubation aus der Körperflüssigkeit, beispielsweise dem Serum, zu entfernen und zu verwerfen sind, werden in dem Verfahren vorzugsweise Goldpartikel mit der Größe von etwa 1 µm eingesetzt, weiter vorzugsweise in einer Menge von 103 - 104 Goldpartikel pro 10 ml. Für diese Anwendung habe sich in der Praxis Behältnisse (z.B. eine Spritze) bewährt, deren Fassungsvolumen etwa 10 ml beträgt.In the variants of the method according to the invention, in which the gold particles are to be removed and discarded after the in vitro incubation from the body fluid, for example the serum, preferably gold particles with the size of about 1 .mu.m, more preferably in one, are used in the method Amount of 10 3 - 10 4 gold particles per 10 ml. In practice, containers (for example a syringe) with a capacity of about 10 ml have proven useful for this application.
In einer Ausführungsform, die sich in der praktischen Anwendung gut bewährt hat, liegen die Goldpartikel in dem Behältnis in einer Menge von 0,3 mg pro 1 ml Körperflüssigkeit vor. Konzentrationen von 0,1 bis 10 mg pro 1 ml sind grundsätzlich geeignet und vorgesehen.In one embodiment that has been well proven in practice, the gold particles are present in the container in an amount of 0.3 mg per 1 ml of body fluid. Concentrations of 0.1 to 10 mg per 1 ml are basically suitable and intended.
Die innere Struktur des Behältnisses ist vorzugsweise frei von Antikoagulantien wie Heparin, Citrat, EDTA oder CPDA, denn im Rahmen der dieser Erfindung zugrunde liegenden Arbeiten wurde überraschenderweise gefunden, dass bei Anwesenheit solcher Substanzen weniger Proteine biosynthetisiert werden als bei Abwesenheit. Insbesondere im Fall von Blut als Körperflüssigkeit und Heparin als Antikoagulanz wurde bei Anwesenheit von Heparin, z.B. als Beschichtung der Behältnisinnenwand, eine erheblich geringere Zytokinproduktion erhalten als bei Abwesenheit von Heparin und anderen Antikoagulantien.The internal structure of the container is preferably free of anticoagulants such as heparin, citrate, EDTA or CPDA, because it has surprisingly been found in the context of the work on which this invention is based that fewer proteins are biosynthesized in the presence of such substances than in the absence. Especially in the case of blood as body fluid and heparin as anticoagulant, in the presence of heparin, e.g. as a coating of the inner wall of the container, a significantly lower cytokine production than in the absence of heparin and other anticoagulants.
Als Behältnis kommt insbesondere eine Spritze in Betracht, weil diese nicht nur als Inkubationsgefäß dienen kann, sondern gleichzeitig auch als Entnahmeinstrument für die Gewinnung der Körperflüssigkeit und/oder als Applikationsinstrument für die Verabreichung der Proteine an einen Patienten geeignet ist.As a container in particular a syringe into consideration, because it can not only serve as Incubationsgefäß, but at the same time as a sampling instrument for obtaining the body fluid and / or as an application instrument for the administration of the proteins to a patient is suitable.
Als Behältnis kommt aber auch ein verschließbarer Beutel in Betracht, weil dieser insbesondere bei größeren Volumina flexibler gehandhabt und gelagert werden kann als eine Spritze mit vergleichbarem Volumen, und weil er technisch einfach und sicher mit einer Spritze gekoppelt werden kann, so dass das Befüllen und Entleeren über diese erfolgen kann.But as a container is also a closable bag into consideration, because it can be handled and stored more flexible especially for larger volumes than a syringe with a comparable volume, and because it can be coupled technically simple and safe with a syringe, so that the filling and emptying can be done over this.
Das offenbarungsgemässe Verfahren eignet sich ganz besonders für die Herstellung (Biosynthese) und Anreicherung von Proteinen aus Blutzellen. Deshalb ist als Körperflüssigkeit insbesondere Blut vorgesehen.The process according to the invention is particularly suitable for the production (biosynthesis) and accumulation of proteins from blood cells. Therefore, in particular blood is provided as body fluid.
Überraschenderweise lässt sich das therapeutisch wirksame Protein Gelsolin besonders gut, d.h. in signifikanten Mengen mit dem erfindungsgemäßen Verfahren herstellen und anreichern. Deshalb ist das offenbarungsgemässe Verfahren insbesondere zur Gewinnung von Gelsolin vorgesehen.Surprisingly, the therapeutically active protein gelsolin is particularly well, i. produce and accumulate in significant amounts with the method according to the invention. Therefore, the process according to the invention is intended in particular for the recovery of gelsolin.
Hinsichtlich der Inkubationsbedingungen für das mit Körperflüssigkeit befüllte Behältnis hat sich in der Praxis gezeigt, dass ein Inkubationszeit von 12 bis 72 Stunden, bevorzugt 24 Stunden, bei einer Temperatur von 20°C bis 41 °C, vorzugsweise 37°, zu guten Ergebnissen führt.With regard to the incubation conditions for the body-filled container, it has been found in practice that an incubation time of 12 to 72 hours, preferably 24 hours, at a temperature of 20 ° C to 41 ° C, preferably 37 °, leads to good results.
Eine Lösung der genannten Aufgabe besteht auch in der Bereitstellung eines Behältnisses für die in-vitro Biosynthese und Anreicherung (In-vitro Induktion) von therapeutisch wirksamen Proteinen in einer Körperflüssigkeit, das sich dadurch auszeichnet, dass das Behältnis Goldpartikel enthält und zur Aufnahme, Lagerung und Wiederabgabe einer Körperflüssigkeitsprobe geeignet ist, und dass es mit einer Hohlnadel (Kanüle, Injektionsnadel) derart koppelbar ist, dass sein Inhalt vermittels dieser Hohlnadel (Kanüle, Injektionsnadel) injiziert werden kann.A solution of the stated object is also to provide a container for in vitro biosynthesis and enrichment (in vitro induction) of therapeutically active proteins in a body fluid, which is characterized in that the container contains gold particles and for receiving, storage and Re-delivery of a body fluid sample is suitable, and that it can be coupled with a hollow needle (cannula, injection needle) such that its contents can be injected by means of this hollow needle (cannula, injection needle).
Mit diesem Behältnis lassen sich insbesondere das zuvor beschriebene Protein-Herstellungsverfahren durchführen und die mit diesem einhergehenden Vorteile nutzen. Die hergestellten Proteine können mittels der ankoppelbaren Hohlnadel direkt an den gewünschten Applikationsort gebracht werden, insbesondere in einen menschlichen oder tierischen Körper eingebracht werden.With this container, in particular, the previously described protein production process can be carried out and use the advantages associated with this. The proteins produced can be brought directly to the desired application site by means of the dockable hollow needle, in particular be introduced into a human or animal body.
Die Goldpartikel weisen vorzugsweise eine definierte Struktur und/oder eine definierte Größe auf. Mikro- und/oder Nanopartikel (Partikelgröße zwischen 10 Nanometern und 500 Mikrometern) sind besonders geeignet. Eine geeignete Menge Goldpartikel in dem Behältnis beträgt 0,1 mg bis 10 mg pro 1 ml Körperflüssigkeit, vorzugsweise 0,3 mg pro 1 ml Körperflüssigkeit.The gold particles preferably have a defined structure and / or a defined size. Micro and / or nanoparticles (particle size between 10 nanometers and 500 micrometers) are particularly suitable. An appropriate amount of gold particles in the container is 0.1 mg to 10 mg per 1 ml of body fluid, preferably 0.3 mg per 1 ml of body fluid.
Bei dem Behältnis handelt es sich vorzugsweise um eine Spritze oder um einen Beutel. Die Spritze hat den Vorteil, dass sie nicht nur als Inkubationsgefäß dienen kann, sondern gleichzeitig auch als Entnahmeinstrument für die Gewinnung der Körperflüssigkeit und/oder als Applikationsinstrument für die Verabreichung der Proteine an einen Patienten. Der Beutel hat den Vorteil, dass er bei größeren Volumina flexibler gehandhabt und gelagert werden kann als eine Spritze mit vergleichbarem Volumen und für das Befüllen und Entleeren mit einer Spritze gekoppelt werden kann.The container is preferably a syringe or a bag. The syringe has the advantage that it can serve not only as an incubation vessel, but at the same time as a sampling device for obtaining the body fluid and / or as an application tool for the administration of the proteins to a patient. The bag has the advantage that it can be handled and stored more flexibly with larger volumes than a syringe of comparable volume and can be coupled with a syringe for filling and emptying.
Als Behältnis eignet sich besonders gut eine handelsübliche Spritze (beispielsweise 5 bis 100 ml Spritzen) ohne besondere Ausgestaltung in ihrem inneren Hohlraum. In den Spritzenzylinder werden die Goldpartikel (z.B. Gold Microcarriers von BioRad Laboratories, Cat. 165-2264) eingebracht.As a container is particularly well a commercial syringe (for example, 5 to 100 ml syringes) without special design in their inner cavity. In the syringe barrels, the gold particles (e.g., Gold Microcarriers from BioRad Laboratories, Cat. 165-2264) are introduced.
Die Spritze, insbesondere die Innenwand des Spritzenzylinders und der in dem Zylinder liegenden Teil des Spritzenkolbens besteht vorzugsweise aus einem Kunststoff, beispielsweise aus Polystyrol, Polyethylen, Polyvinylchlorid, Polypropylen (neutrale S-Monovetten, Sarstedt) oder einem ähnlichen Stoff oder Gemischen davon.The syringe, in particular the inner wall of the syringe barrel and the syringe barrel located in the cylinder, is preferably made of a plastic, for example polystyrene, polyethylene, polyvinyl chloride, polypropylene (neutral S-Monovetten, Sarstedt) or a similar substance or mixtures thereof.
Das Behältnis eignet sich besonders gut für die Herstellung (Biosynthese) und Anreicherung von Proteinen, insbesondere von Gelsolin, aus Blutzellen. Deshalb ist als Körperflüssigkeit insbesondere Blut vorgesehen.The container is particularly well suited for the production (biosynthesis) and enrichment of proteins, in particular of gelsolin, from blood cells. Therefore, in particular blood is provided as body fluid.
Die mit dem offenbarungsgemässen Verfahren in einer Körperflüssigkeit hergestellten Proteine können zusammen mit der Körperflüssigkeit und den Goldpartikeln oder ohne die Goldpartikel als Arzneimittel zur Behandlung von Krankheiten eingesetzt werden.The proteins prepared by the method according to the invention in a body fluid can be used together with the body fluid and the gold particles or without the gold particles as a medicament for the treatment of diseases.
Die mit den offenbarungsgemässen Verfahren hergestellten proteinangereicherten und insbesondere Zytokinin- und Gelsolinangereicherten Körperflüssigkeiten, insbesondere Blutseren, stellen eine sichere, kostengünstig und schnell herzustellende und besonders nebenwirkungsarme Alternative zu vergleichbaren konventionellen Arzneimittelpräparaten dar.The protein-enriched and, in particular, cytokinin-enriched and gelsolin-enriched body fluids, in particular blood sera, which are produced using the methods according to the invention, represent a safe, cost-effective and rapidly produced alternative with low side effects compared to comparable conventional pharmaceutical preparations.
Die vorliegende Erfindung betrifft ein Zytokinin- und Gelsolinangereichertes Blut, zur Verwendung als Arzneimittel bzw. zur Herstellung eines Arzneimittels, erhältlich dadurch, dass Blut, in ein Behältnis aufgenommen wird, welches Goldpartikel enthält, vorzugsweise Goldpartikel mit der Größe von etwa 1 µm und vorzugsweise in einer Menge von 103 - 104 Goldpartikel pro 10 ml Behältnis (oder 0.3 mg Goldpartikel mit einem Durchmesser 1 µm pro 1 ml Blut/Behältnis), dass diese Mischung aus Blut, und Goldpartikeln inkubiert wird (beispielsweise über 12-72 Stunden, vorzugsweise über 24 Stunden und bei 20°C bis 41 °C, vorzugsweise bei 37°C), und dass anschließend die Goldpartikel und vorzugsweise (d.h. optional) zudem Blutzellen und andere unlösliche Bestandteile aus der Körperflüssigkeit, insbesondere dem Blut, entfernt und verworfen werden (vorzugsweise durch Zentrifugation und/oder Sterilfiltration).The present invention relates to a cytokinin and gelatin-enriched blood for use as a medicament or for the preparation of a medicament obtainable by taking blood into a container containing gold particles, preferably gold particles of about 1 μm in size, and preferably in a quantity of 10 3 - 10 4 gold particles per 10 ml container (or 0.3 mg of gold particles with a diameter of 1 μm per 1 ml of blood / container) that this mixture of blood, and gold particles is incubated (for example over 12-72 hours, preferably over 24 hours and at 20 ° C to 41 ° C, preferably at 37 ° C) and then that the gold particles and preferably (ie optionally) also blood cells and other insoluble constituents from the body fluid, especially the blood, are removed and discarded ( preferably by centrifugation and / or sterile filtration).
Die Offenbarung betrifft außerdem die Verwendung von autologem oder homologem Blut und Goldpartikeln in Kombination als Arzneimittel bzw. zur Herstellung eines Arzneimittels. Mit anderen Worten: Gegenstand vorliegender Erfindung ist auch eine Stoffmischung aus autologem oder homologem Blut und Goldpartikeln zur Verwendung als Arzneimittel, bzw. ein Arzneimittel umfassend die Stoffmischung autologes oder homologes Blut und Goldpartikeln inklusive der darin angereicherten Proteine als Wirkstoffkombination.The disclosure also relates to the use of autologous or homologous blood and gold particles in combination as a pharmaceutical or for the manufacture of a medicament. In other words, subject of the present invention is also a mixture of autologous or homologous blood and gold particles for use as drugs, or a drug comprising the mixture autologous or homologous blood and gold particles including the proteins enriched therein as a combination of active ingredients.
Die offenbarten Arzneimittel ermöglichen eine besonders einfache, kostengünstige, risikoarme, und effektive Behandlung.The disclosed pharmaceuticals enable a particularly simple, inexpensive, low-risk, and effective treatment.
Die beschriebenen Arzneimittel eignen sich insbesondere für die Behandlung von degenerativen Gewebeerkrankungen, vor allem von Arthrosen und Tendinosen. Vor allem das mit Goldpartikeln inkubierte und anschließend von Partikeln befreite Arzneimittel ist für die Behandlung von Arthrose und anderen Krankheiten, die mit Gewebedegenerationen assoziiert sind, und/oder die mit einem Gelsolinmangel assoziiert sind, gut geeignet und dafür vorgesehen.The medicaments described are particularly suitable for the treatment of degenerative tissue diseases, especially of arthroses and tendinoses. In particular, the drug-incubated and subsequently particle-depleted drug is well-suited and contemplated for the treatment of osteoarthritis and other diseases associated with tissue degeneration and / or associated with gelsolin deficiency.
Eine besondere Ausführungsform der erfindungsgemäßen Arzneimittel zeichnet sich dadurch aus, dass die mit Goldpartikeln inkubiertes Blut nach der Inkubation einen Gehalt an Gelsolin aufweist, der wenigstens das Doppelte des entsprechenden Blutnormwerts für Gelsolin beträgt. Der Begriff "entsprechender Blutnormwert für Gelsolin" steht im vorliegenden Kontext für den humanmedizinischen oder tiermedizinischen Normwert für Gelsolin im Blut derjenigen Patientengruppe, die mit dem Arzneimittel behandelt werden soll. Die Patientengruppe ist dabei charakterisiert durch ihre zoologische Art- und Rassenzugehörigkeit, Geschlecht und Alter.A particular embodiment of the medicaments according to the invention is characterized in that the blood incubated with gold particles after incubation has a content of gelsolin which is at least twice the corresponding standard blood level for gelsolin. The term "corresponding standard blood level for gelsolin" in the present context stands for the human medical or veterinary standard value for gelsolin in the blood of the group of patients to be treated with the drug. The patient group is characterized by its zoological species and race, sex and age.
Dieses Gelsolin-reiche Arzneimittel ist für die Behandlung von Arthrose vorgesehen, die mit einem Gelsolinmangel im Blut des Patienten einhergeht. Das inkubierte Blut-Gold-Gemisch kann als Ganzes oder in Teilen appliziert werden. Bei Bedarf kann es vor der Verabreichung an den (tierischen oder menschlichen) Patienten noch einer Zentrifugation und/oder Sterilfiltration unterworfen werden, um z.B. Zellen und Zellfragmente aus dem Blut zu entfernen und damit gleichzeitig auch das Injektionsvolumen zu verringern.This Gelsolin-rich medicine is intended for the treatment of osteoarthritis associated with a lack of gelsolin in the patient's blood. The incubated blood-gold mixture can be administered in whole or in part. If necessary, it may be subjected to centrifugation and / or sterile filtration prior to administration to the (animal or human) patient, e.g. To remove cells and cell fragments from the blood and at the same time to reduce the injection volume.
Die Erfindung wird im folgenden anhand von Ausführungsbeispielen und dazugehörigen Figuren näher erläutert:
Figur 1 zeigt:- eine MID-FTIR-Spektroskopie-Analyse des Proteinprofils in Blutseren verschiedener Patienten vor (T0) und nach (T24) Durchführung des offenbarten Verfahrens im Vergleich zu Kontrollen. BLAU repräsentiert das Proteinprofil für den Zeitpunkt T0, GRÜN zeigt das Proteinprofil der mit dem erfindungsgemäßen Verfahren behandelten Proben zum Zeitpunkt T24, ROT zeigt das Proteinprofil der Kontrollseren zum Zeitpunkt T24.
Figur 2 zeigt:- die Ergebnisse einer Multiplex-Analyse der Proteine:
GS = Gelsolin
IL-4 = Interleukin-4
IL-10 = Interleukin-10
IL-13 = Interleukin-13
IL-1Ra = Interleukin-1 Rezeptorantagonist
IL-1ß = Interleukin-1ß
TNF-α = Tumornekrasefaktor alpha
G-CSF = Granulocyte-Colony Stimulating factor
GM-CSF = Granulocyte-Macrophage Colony Stimulating factor
IFN-g = Interferon Gamma
SCGF-ß = Hematopoietic Stem cell growth factor
MIP-1a = Macrophage inflammatory protein-1a
MIP-1ß = Macrophage inflammatory protein-1ß
VEGF = Vascular endothelial growth factor
IL-18 = Interleukin-18
MCP-3 = Macrophage chemotactic protein
SDF-a = Stromal derived factor
basic FGF = Fibroblast growth factor
GROa = Growth-regulated oncogene alpha in Blutproben zum Zeitpunkt T0 ("T0") und nach 24 Stunden Inkubation einerseits ohne weitere Behandlung ("Kontrolle"), und anderseits mit Behandlung entweder gemäßWO 9909051 Figur 3 zeigt:- die graphische Dokumentation des Schwellungsgrades aller untersuchten Pferde vor bzw. nach der erfindungsgemäßen Behandlung zu den jeweiligen Nachuntersuchungszeitpunkten
Figur 4 zeigt:- die graphische Dokumentation der Lahmheit aller untersuchten Pferde vor bzw. nach der erfindungsgemäßen Behandlung zu den jeweiligen Nachuntersuchungszeitpunkten
Figur 5 zeigt:- KOOS-Score vor und nach der Behandlung bei Patienten mit Gonarthrose
Figur 6 zeigt:- Ergussbildung und Gelsolinkonzentration in der Synovialflüssigkeit bei einer Patientin mit Gonarthrose im Verlauf: nach der 1. (T1), 2. (T2)
bzw 3. Injektion (T3) Figur 7 zeigt:- die Proteoglykanfreisetzung von Explantaten/Knorpel-Knochen-Präparaten nach Stoßbehandlung.
- FIG. 1 shows:
- an MID-FTIR spectroscopic analysis of the protein profile in blood sera of different patients before (T0) and after (T24) performing the disclosed method compared to controls. BLUE represents the protein profile for the time T0, GREEN shows the protein profile of the samples treated by the method according to the invention at time T24, ROT shows the protein profile of the control sera at time T24.
- FIG. 2 shows:
- the results of a multiplex analysis of the proteins:
GS = gelsolin
IL-4 = interleukin-4
IL-10 = interleukin-10
IL-13 = interleukin-13
IL-1Ra = Interleukin-1 receptor antagonist
IL-1β = interleukin-1β
TNF-α = Tumor Nasal Factor alpha
G-CSF = granulocyte colony stimulating factor
GM-CSF = granulocyte-macrophage colony stimulating factor
IFN-g = interferon gamma
SCGF-ß = hematopoietic stem cell growth factor
MIP-1a = macrophage inflammatory protein-1a
MIP-1β = macrophage inflammatory protein-1β
VEGF = vascular endothelial growth factor
IL-18 = interleukin-18
MCP-3 = macrophage chemotactic protein
SDF-a = stromal derived factor
basic FGF = fibroblast growth factor
GROa = growth-regulated oncogene alpha in blood samples at time T0 ("T0") and after 24 hours incubation on the one hand without further treatment ("control"), and on the other hand with treatment either according toWO 9909051 - FIG. 3 shows:
- the graphic documentation of the degree of swelling of all examined horses before and after the treatment according to the invention at the respective follow-up times
- FIG. 4 shows:
- the graphic documentation of the lameness of all examined horses before or after the treatment according to the invention at the respective follow-up times
- FIG. 5 shows:
- KOOS score before and after treatment in patients with gonarthrosis
- FIG. 6 shows:
- Effusion and concentration of gelsolin in the synovial fluid in a patient with gonarthrosis in the course: after the 1st (T1), 2nd (T2) or 3rd injection (T3)
- FIG. 7 shows:
- the proteoglycan release of explant / cartilage-bone preparations after impact treatment.
Jeweils zwei Blutproben der Menge 9 ml von 11 verschiedenen Patienten (Nr. 1-11) wurden in ein offenbartes Behältnis, nämlich eine zuvor mit 2,7 mg Goldpartikeln (mit einem Durchmesser von 1 µm) befüllte 9ml Spritze aufgenommen. Eine gleiche Menge Blutprobe desselben Ursprungs (Patienten) wurde in eine gleichartige 9ml Spritze ohne Gehalt an Goldpartikeln aufgenommen. Die Proben wurden zu unterschiedlichen Zeitpunkten analysiert, nämlich zum Zeitpunkt T0 sofort nach der Blutentnahme und zum Zeitpunkt T24 nach Inkubation für 24 Stunden bei 37°C.Two 9 ml blood samples each from 11 different patients (Nos. 1-11) were taken into a disclosed container, namely a 9 ml syringe previously filled with 2.7 mg of gold particles (with a diameter of 1 μm). An equal amount of blood sample of the same origin (patient) was taken up in a similar 9 ml syringe without gold particle content. The samples were analyzed at different time points, namely at time T0 immediately after blood collection and at time T24 after incubation for 24 hours at 37 ° C.
Mittels Fourier-Transformations-Infrarot-Spektroskopie im mittleren Infrarot-Bereich (Spektralspektrum von 4000 cm-1 bis 400 cm-1), kurz MID-FTIR-Spektroskopie (z.B. dem AquaSpec-Verfahren der Firma Micro-Biolytics GmbH/Esslingen) wurden die Proben analysiert.Using Fourier transform infrared spectroscopy in the mid-infrared range (spectral spectrum from 4000 cm -1 to 400 cm -1 ), MID-FTIR spectroscopy (eg the AquaSpec method of Micro-Biolytics GmbH / Esslingen), the samples were analyzed.
Das Messprinzip der Fourier-Transformations-Infrarot-Spektroskopie) beruht auf der Bestrahlung eines Stoffes mit elektromagnetischen Wellen, wobei bestimmte Frequenzbereiche absorbiert werden. Da Infrarotstrahlung energetisch im Bereich der Schwingungsniveaus von Molekülbindungen liegt, führt die Absorption zu einer Schwingungsanregung der Bindungen. Diese wird in Form von Ausschlägen im gemessenen Spektrum (Diagramm) sichtbar. Da die dazu notwendigen Energien bzw. Frequenzen charakteristisch für die jeweiligen Bindungen sind, können so auch Materialien identifiziert und Strukturen aufgeklärt werden.The measuring principle of Fourier transform infrared spectroscopy) is based on the irradiation of a substance with electromagnetic waves, whereby certain frequency ranges are absorbed. Since infrared radiation is energetically in the range of vibrational levels of molecular bonds, absorption leads to vibrational excitation of the bonds. This is visible in the form of deflections in the measured spectrum (diagram). Since the energies or frequencies necessary for this are characteristic of the respective bonds, materials can also be identified and structures can be elucidated.
Die FTIR-Spektroskopie ist insbesondere für die Analyse struktureller, reaktionsinduzierter Veränderungen in einem biologischen Makromolekül geeignet ist. Mit ihr lassen sich biologische Systeme, insbesondere proteinhaltige wässrige Flüssigkeiten untersuchen. Die Probe wird dabei weder verändert noch zerstört und es kann unter nativen Bedingungen des Biomoleküls gearbeitet werden, . d.h. es kann der "Ist-Zustand" gemessen werden, weil weder eine Fixierung noch ein andersartige Aufbereitung der Proben notwendig ist.The FTIR spectroscopy is particularly suitable for the analysis of structural, reaction-induced changes in a biological macromolecule. It can be used to study biological systems, in particular protein-containing aqueous liquids. The sample is neither altered nor destroyed and it can be used under native conditions of the biomolecule,. i.e. It is possible to measure the "actual state" because neither a fixation nor a different preparation of the samples is necessary.
Da alle molekularen Bestandteile des Proteins Absorptionsbanden im infraroten Spektralbereich besitzen, können nahezu alle Bereiche eines Proteins beobachtet und detaillierte Informationen über die Struktur des Proteins erhalten werden.Since all molecular components of the protein have absorption bands in the infrared spectral range, almost all regions of a protein can be observed and detailed information about the structure of the protein can be obtained.
Das MID-FTIR-Spektroskopie-Verfahren ermöglicht die automatisiert und reproduzierbar Erkennung und Quantifizierung von Änderungen der Protein-Konformation und die Bestimmung der Proteinkonzentration. Es können bereits sehr geringe Proteinkonzentrationen (bis unter 0,1 mg/ml) und geringste Konformations-Änderungen detektiert werden.The MID-FTIR spectroscopy method allows automated and reproducible detection and quantification of changes in protein conformation and determination of protein concentration. It is already possible to detect very low protein concentrations (below 0.1 mg / ml) and lowest conformational changes.
Bei der Durchführung des Verfahrens im Zuge der vorliegenden Erfindung wurden die Blutproben mit und ohne Goldpartikel im "Ist"-Zustand in einer hoch präzisen biokompatiblen und für wässrige Proben geeigneten spektroskopischen Messzelle (Transmissionszelle) analysiert. Unter Verwendung interner Kalibrationen wurde von jeder vermessenen Probe automatisch die Konzentration des gelösten Proteins und dessen Sekundärstruktur (alpha-Helix, beta-Faltblatt) bestimmt.In carrying out the method in the present invention, the blood samples were analyzed with and without gold particles in the "as is" state in a highly accurate biocompatible and suitable for aqueous samples spectroscopic measuring cell (transmission cell). Using internal calibrations, the concentration of the dissolved protein and its secondary structure (alpha-helix, beta-sheet) were automatically determined from each measured sample.
Die Ergebnisse dieser Messung sind in
Mit einem Multiparameter-Analyseverfahren (Synonym: Multiplex-Analyse) auf der Basis von unterscheidbar codierten Mikropartikeln (z.B. dem BioPlex™2200-System der Firma BioRad Laboratories, München) wurden folgende Proteine in den Proben mit dem erfindungsgemäßen Verfahren ("Erfndung"), dem Verfahren gemäß
- Gelsolin (GS), Interleukin-4 (IL-4), Interleukin-10 (IL-10), Interleukin-13 (IL-13), Interleukin-1 Rezeptorantagonist (IL-1Ra), Interleukin-1ß (IL-1ß), Tumornekrosefaktor alpha (TNF-a), Granulocyte-Colony Stimulating factor (G-CSF), Granulocyte-Macrophage Colony Stimulating factor (GM-CSF), Interferon Gamma (IFN-g), Hematopoietic Stem cell growth factor (SCGF-ß), Macrophage inflammatory protein-1a (MIP-1a), Macrophage inflammatory protein-1ß (MIP-1ß), Vascular endothelial growth factor (VEGF), Interleukin-18 (IL-18), Macrophage chemotactic protein (MCP-3), Stromal derived factor (SDF-a), Fibroblast growth factor basic (FGF), Growth-regulated oncogene alpha (GROa).
- Gelsolin (GS), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-1 receptor antagonist (IL-1Ra), interleukin-1ß (IL-1ß) , Tumor Necrosis Factor Alpha (TNF-a), Granulocyte Colony Stimulating Factor (G-CSF), Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), Interferon Gamma (IFN-g), Hematopoietic Stem Cell Growth Factor (SCGF-ß) Macrophage inflammatory protein-1a (MIP-1a), macrophage inflammatory protein-1β (MIP-1β), vascular endothelial growth factor (VEGF), interleukin-18 (IL-18), macrophage chemotactic protein (MCP-3), stromal derived factor (SDF-a), fibroblast growth factor basic (FGF), growth-regulated oncogenic alpha (GROa).
Diese Proteine spielen bei der Gewebedegeneration und der Gewebereparation wichtige Rollen.These proteins play important roles in tissue degeneration and tissue repair.
Die Ergebnisse dieser Analyse sind in Tabelle 1 und graphisch in
Sie zeigen, dass bei den Proben ("Erfindung") nach 24 Stunden eine erhebliche Zunahme der Gelsolinkonzentration (Faktor 10) stattgefunden hat, während bei den Vergleichsproben ("Kontrolle-T24" und "StdT-T24") keine Gelsolinanreicherung sondern eher ein Gelsonlinschwund festgestellt wurde. Auch die Konzentrationen von Tumornekrosefaktor alpha (TNF-a), Macrophage chemotactic protein (MCP-3) und Growth-regulated oncogene alpha ("GROa") waren in den erfindungsgemäß behandelten Proben ("Erfindung") wesentlich (TNF-a: Faktor 30-40, MCP-3: Faktor 20-30) oder zumindest deutlich (GROa: Faktor 2) höher als in den Vergleichsproben ("Kontrolle-T24" und "StdT-T24").They show that the samples (" invention ") had a significant increase in the gelsolin concentration (factor 10) after 24 hours, while the control samples (" control T24 " and " StdT-T24 ") did not show gelatinization but rather gelsonline depletion was determined. The concentrations of tumor necrosis factor alpha (TNF-α), macrophage chemotactic protein (MCP-3) and growth-regulated oncogenic alpha ("GROα") were also significant in the samples treated according to the invention ("invention") (TNF-a: factor 30 -40, MCP-3: factor 20-30) or at least significantly (GROa: factor 2) higher than in the control samples ("Control-T24" and "StdT-T24").
Im Rahmen einer prospektiven klinischen Studie wurden 8 Pferde mit einer ausgeprägten Weichteilschwellung aufgrund von Tendinosen (degenerative Veränderungen an Sehnen im Bereich der Knochenansätze) mit dem erfindungsgemäßen Arzneimittel behandelt.In a prospective clinical study, 8 horses with pronounced soft tissue swelling due to tendinosis (degenerative changes to tendons in the area of the bone attachment) were treated with the drug according to the invention.
Für die Herstellung des Arzneimittels wurde ein Behältnis in Form einer Goldpartikel enthaltenden Spritze mit Blut des betreffenden Tieres gefüllt und 24 Stunden bei einer Temperatur im Bereich von 37°C inkubiert. Nach Ablauf der Inkubationszeit war das Arzneimittel in Gestalt des sich in der Spritze befindenden Blutes mit den zwischenzeitlich synthetisierten und angereicherten Proteinen, insbesondere Zytokinen und Gelsolin, sowie den Goldpartikeln fertig gestellt und konnte direkt und unmittelbar eingesetzt werden.For the preparation of the drug, a container in the form of a gold particle-containing syringe was filled with blood of the animal in question and incubated for 24 hours at a temperature in the range of 37 ° C. After the incubation period, the drug in the form of the blood in the syringe with the synthesized and enriched in the meantime proteins, especially cytokines and gelsolin, and the gold particles was completed and could be used directly and immediately.
Da das Arzneimittel in einer Spritze hergestellt wurde, konnte es ohne Umfüllung und damit ohne die Risiken von Kontamination und Materialverlust dem betreffenden Tier per Injektion verabreicht werden.Because the drug was manufactured in a syringe, it could be given by injection to the animal in question without being refilled and thus without the risks of contamination and material loss.
Für die vorliegende Studie wurden zum Zeitpukt T0 für jedes Pferd vier derartige Arzneimittel-Dosen hergestellt, d.h. es wurden vier Goldpartikel enthaltende Spritzen mit Blut des betreffenden Tieres gefüllt und 24 Stunden bei einer Temperatur im Bereich von 37°C inkubiert.For the present study, four such drug doses were prepared for each horse at time T0, ie, syringes containing four gold particles were filled with the animal's blood and incubated at a temperature in the range of 37 ° C for 24 hours.
Diese Arzneimittel-Injektionen wurden dem jeweiligen Pferd im Abstand von jeweils einer Woche verabreicht. Die erste Verabreichung erfolgte zum Zeitpunkt T24, die zweite, dritte und vierte nach Woche 1, nach Woche 2 und nach Woche 3. Die Arzneimittel-haltigen Spritzen für die zweite, dritte und vierte Applikation wurden bis zu ihrer Verwendung bei minus 20°C gelagert.These drug injections were given to the respective horse one week apart. The first administration was at time T24, the second, third and fourth after
Der Schwellungszustand wurde nach 1 Woche, 2 Wochen, 3 Wochen, 3 Monaten, 6 Monaten und 1 Jahr überprüft und anhand einer Skala von 0-5 (0 = keinerlei Schwellung, 5 = massive Schwellung) bewertet.The state of swelling was checked after 1 week, 2 weeks, 3 weeks, 3 months, 6 months and 1 year and scored on a scale of 0-5 (0 = no swelling, 5 = massive swelling).
In allen 8 Fällen wurde bereits nach 3 Wochen eine signifikante Reduktion der Schwellung festgestellt. Nach 6 Monaten und auch nach einem Jahr waren alle Pferde komplett schwellungsfrei. Es wurden im Rahmen der Behandlungen keinerlei Nebenwirkungen festgestellt.In all 8 cases, a significant reduction of the swelling was already detected after 3 weeks. After 6 months and also after one year, all horses were completely free of swelling. No side effects were noted during the treatment.
Die Ergebnisse dieser Studie sind in
In einer weiteren Pferdestudie wurden 11 Pferde mit dem klinischen Krankheitssymptom der Lahmheit (12 betroffene Extremitäten) mit dem erfindungsgemäßen Arzneimittel behandelt. Die medizinischen Ursachen für die Lahmheiten waren in sechs Fällen degenerative Knorpelveränderungen (n=6) in/an Gelenken und in sechs Fällen Weichteilerkrankungen (n=6).In another
Für die Herstellung des Arzneimittels wurden wiederum pro Pferd vier Behältnisse in Form Goldpartikel enthaltender Spritzen mit Blut des betreffenden Tieres gefüllt und 24 Stunden bei einer Temperatur im Bereich von 37°C inkubiert. Nach Ablauf der Inkubationszeit war das Arzneimittel in Gestalt des sich in der Spritze befindenden Blutes mit den zwischenzeitlich synthetisierten und angereicherten Proteinen, insbesondere Zytokinen und Gelsolin, sowie den Goldpartikeln im Prinzip fertig gestellt. Um das Injektionsvolumen zu minimieren wurden in einem anschließenden Zentrifugationsverfahren korpuskuläre Anteile durch Zentrifugation über 10 Minuten bei 5000 U/min entfernt. Nur die jeweiligen Überstandes wurden für die Injektionen eingesetzt.For the preparation of the drug, four containers in the form of syringes containing gold particles per horse were again filled with blood of the relevant animal and incubated for 24 hours at a temperature in the region of 37 ° C. At the end of the incubation period, the drug was in principle finished in the form of the blood in the syringe with the proteins synthesized and enriched in the meantime, in particular cytokines and gelsolin, and the gold particles. In order to minimize the injection volume, corpuscular portions were added by centrifugation for 10 minutes in a subsequent centrifugation procedure 5000rpm away. Only the respective supernatants were used for the injections.
Diese Arzneimittel-Injektionen wurden dem jeweiligen Pferd im Abstand von jeweils einer Woche verabreicht. Die erste Verabreichung erfolgte zum Zeitpunkt T24, die zweite, dritte und vierte nach Woche 1, nach Woche 2 und nach Woche 3. Die Arzneimittel-haltigen Spritzen für die zweite, dritte und vierte Applikation wurden bis zu ihrer Verwendung bei minus 20°C gelagert.These drug injections were given to the respective horse one week apart. The first administration was at time T24, the second, third and fourth after
Die Lahmheit wurde nach 1, 2 und 3 Wochen, 3 und 6 Monaten und 1 Jahr überprüft und der Lahmheitsgrad wurde anhand einer Skala von 0-4 (0 = keinerlei Lahmheit, 5 = massive Lahmheit) gemäß AAEP = "American Association of Equine Practitioners" bewertet.Lameness was checked at 1, 2 and 3 weeks, 3 and 6 months and 1 year and lameness was graded on a scale of 0-4 (0 = no lameness, 5 = massive lameness) according to AAEP = American Association of Equine Practitioners " rated.
In allen 12 Fällen wurde bereits nach 3 Wochen eine signifikante Reduktion der Lahmheit festgestellt. Nach 6 Monaten und auch nach einem Jahr waren alle Pferde komplett symptomfrei, insbesondere lahmfrei. Es wurden im Rahmen der Behandlungen keinerlei Nebenwirkungen festgestellt.In all 12 cases, a significant reduction in lameness was noted after 3 weeks. After 6 months and also after one year, all horses were completely symptom free, especially lame free. No side effects were noted during the treatment.
Die Ergebnisse dieser Studie sind in
Als Goldpartikel wurde Goldpuder (Partikelgröße 1 µm, Firma Bio-Rad Laboratories, München) verwendet. Die Goldpartikel wurden zunächst sterilisiert, wie vom Hersteller empfohlen: Die benötigte Menge Goldpartikel/Goldpuder, beispielsweise 30 mg, wurde mit 1 ml 70 %igen Ethanol versetzt, 10 Minuten unter leichtem Rühren (Mischer, Vortexer) inkubiert, nach 1 Minute absetzen kurz zentrifugiert und anschließend der Überstand entfernt. Danach wurden die Goldpartikel dreimal mit je 1 ml sterilem Aqua bidest. gewaschen. Nach dem letzten Waschgang mit abschließender Zentrifugation und Abschütten des Überstands wurden die Goldpartikel in sterilem PBS resuspendiert und auf die gewünschte Konzentration, z.B. 60 mg/ml), eingestellt. Unter ständigem Schütteln wurden mit einer Pipette 10µl der Goldpartikellösung entnommen und in eine S-Monovette - neutral/9ml (92 x 16 mm) von Sarstedt (REF 02.1726.001) überführt. Die Monovette wurde zunächst in einer sterilen Werkbank geöffnet (Schraubverschluss), die 10µl Goldpartikellösung auf die innere Spritzenwand appliziert und anschließend wieder verschlossen. Die befüllten Monovetten lagerten bis zur ihrer Verwendung bei Raumtemperatur. Für den Einsatz bei der Durchführung des offenbarten Verfahrens wurden in die derart vorbereiteten Monovetten 9 ml Körperflüssigkeit (z.B. Blut) aufgezogen und mit der Goldpartikel/PBS Lösung gemischt.The gold particles used were gold powder (
Im Rahmen einer prospektiven Längsschnittstudie wurden durch einen zugelassenen Arzt insgesamt 9 Patienten bzw. 13 Gelenke mit röntgenologisch nachgewiesener Gonarthrose behandelt. Der Arthrosegrad war nach Kellgren-Lawrence-Klassifkation (
Die Auswertung des KOOS-Score hinsichtlich der Parameter 'Symptome' und 'Sportaktivität' zeigte nach 3 und. 6 Monaten eine deutliche Verbesserung der klinischen Symptomatik (vgl.
Bei einer Patientin zeigte sich initial eine erhebliche Ergussbildung. Der Erguss wurde vor der Injektions-Behandlung jeweils abpunktiert, hinsichtlich der Ergussmenge dokumentiert und die Synoviapunktate hinsichtlich der Gelsolinkonzentration untersucht. Da die Gelsolinkonzentration vom Verdünnungsgrad der Ergussbildung abhängig ist, wurde die Gelsolinkonzentration auf die Urea-Konzentration bezogen (
Durch die intraartikuläre Applikation des hergestellten Serums, d.h. des Bluts nach Inkubation mit Goldpartikeln und anschließender Entfernung der (aller) Partikel konnte die intraartikuläre Gelsolinkonzentration deutlich gesteigert werden (vgl.
Diese Studie belegt somit die Verwendung des Arzneimittels zur Behandlung von Arthrose.This study thus demonstrates the use of the drug for the treatment of osteoarthritis.
Der Einfluss mechanischer Überlastung auf den Gelenkknorpel im Rahmen der Arthroseentstehung ist hinlänglich bekannt. Es existieren auch gut etablierte und validierte in-vitro Modelle, die den Einfluss mechanischer Belastung bei Knorpel-Knochenexplantaten untersuchen, z.B. das Modell von Huser und Davies (
Im Verlauf der Untersuchungen, die zu der vorliegenden Erfindung geführt haben, wurde in einer tierexperimentellen Studie am Minischwein "Göttinger Minipig" bei 6 Tieren mit 9 ml Spritzen, die jeweils 103 Goldpartikel enthielten, pro Tier jeweils 4 Proben (à 9 ml) Blut entnommen und gemäß dem erfindungsgemäßen Verfahren 24 h bei 37°C inkubiert. Im Anschluß an die Inkubationszeit wurden die Blutproben in den Spritzenzylindern zentrifugiert und je Spritze (bzw. Spritzenzylinder) der Überstand durch einen Sterilfilter in einen frischen/neuen goldpartikelfreien Spritzenzylinder überführt.In the course of the investigations which led to the present invention, in an animal study on minipigs "Göttinger Minipig" in each of 6 animals with 9 ml syringes, each containing 10 3 gold particles, 4 samples (9 ml each) of blood per animal taken and incubated according to the method of the invention for 24 h at 37 ° C. Following the incubation period, the blood samples were taken in the syringe barrels centrifuged and per syringe (or syringe barrel) the supernatant through a sterile filter in a fresh / new gold particle-free syringe barrel transferred.
Parallel zur Entnahme der Blutproben wurden den der betreffenden Tieren unter aseptischen Bedingungen Knorpel-Knochen-Proben aus den jeweiligen Hüftköpfen entnommen und zu 8x8x10 mm (Länge/Breite/Höhe) Blöcken zugeschnitten. Der Knorpel war bei allen Explantaten makroskopisch intakt.In parallel with the blood samples, the cartilage-bone samples from the respective femoral heads were removed from the respective animals under aseptic conditions and cut to 8x8x10 mm (length / width / height) blocks. The cartilage was macroscopically intact in all explants.
Bis zum Einsatz in dem Stoßbelastungstest (Impaktierungstest) wurden Knorpel-Knochen-Probenblöcke in DMEM-Medium mit dem Zusatz von 10 % Humanem Serum (HS), 100 U/ml Pencillin, 100 µg/ml Gentamycin und 1,25 U/ml Amphotericin B aufbewahrt.Until used in the impact load test, cartilage-bone sample blocks were placed in DMEM medium supplemented with 10% human serum (HS), 100 U / ml pencillin, 100 μg / ml gentamycin, and 1.25 U / ml amphotericin B stored.
Noch am Tage der Probenentnahme wurde der Stoßbelastungstest (Impaktierungstest) durchgeführt. Dazu wurde jeweils ein Knorpel-Knochen-Probenblock/Explantat in einem zylinderförmigen Freifallturm ("drop tower") aus Polymethylmethacrylate mit den Maßen 33 cm Höhe und 4 cm axial mit Abstand unterhalb eines Stoßkolbens unter sterilen Bedingungen angeordnet. Der Stoßkolben hatte die Form eines Zylinders mit den Maßen 5 cm Höhe und 3,94 cm Durchmesser, und sein Gewicht betrug 493 g. Der Stoßkolben war über ein Gewinde mit der eigentlichen Stoßscheibe ("Impaktorstück") verbunden, die ein Gewicht von 7 g, eine Höhe/Dicke von 1 cm und einen Durchmesser von 0,6 cm aufwies. Die Stoßbelastung/Impaktierung erfolgte einmalig pro Knorpel-Knochen-Probenblock im "drop tower" durch den freien Fall des Stoßkolbens mit Stoßscheibe (Impaktors) aus 15 cm Höhe unter auf die Probe. Die Stoßbelastung betrug dabei 0.736 J auf eine Knorpeloberfläche von 28.3 mm2.The impact load test (impaction test) was carried out on the day of the sampling. For this purpose, in each case a cartilage-bone sample block / explant was arranged in a cylindrical drop tower made of polymethyl methacrylate with the dimensions 33 cm height and 4 cm axially with distance below an impact piston under sterile conditions. The butt piston had the shape of a cylinder measuring 5 cm high and 3.94 cm in diameter and weighing 493 g. The butt piston was threadedly connected to the actual impact disk ("impactor piece"), which had a weight of 7 g, a height / thickness of 1 cm and a diameter of 0.6 cm. The impact loading / impacting took place once per cartilage-bone sample block in the "drop tower" by the free fall of the impact piston with impact disk from 15 cm height below the sample. The impact load was 0.736 J on a cartilage surface of 28.3 mm 2 .
Die Knorpel-Knochen-Probenblöcke/Explantate wurden in 3 Gruppen unterteilt:
- Explantate ohne Stoßbehandlung = "nicht-impaktierte Kontrollen" = "0-Kontrollen")
- Explantate mit Stoßbehandlung = "impaktierte Kontrollen" = "Stoß-Kontrollen"
- Explantate mit Stoßbehandlung und anschließender Inkubation mit dem erfindungsgemäß hergestellten, proteinangereichtem Blutserum
- Explants without shock treatment = "non-impacted controls" = "0 controls")
- Shock treatment explants = "impacted controls" = "push controls"
- Explants with shock treatment and subsequent incubation with the protein-rich blood serum prepared according to the invention
Im Anschluß an die Stoßbehandlung wurden die behandelten Knochen-Knorpel-Präparate und ebenso die 0-Kontrollen 3 mal mit PBS gewaschen und in 12-well Kulturplatten überführt. Jedes Explantat wurde mit 3 ml des jeweiligen Behandlungsmediums angesetzt und unter standardisierten Bedingungen (37°C, 5 % CO2,) im Brutschrank inkubiert. Das Kulturmedium wurde alle 72 Stunden gewechselt.Following the shock treatment, the treated bone-cartilage preparations as well as the 0 controls were washed 3 times with PBS and in 12-well culture plates transferred. Each explant was prepared with 3 ml of the respective treatment medium and incubated under standardized conditions (37 ° C, 5% CO 2 ,) in the incubator. The culture medium was changed every 72 hours.
Das offenbarungsgemäss hergestellte proteinangereichte Blutserum wurde bei den Explantat der betreffenden Explantat-Gruppe am Tag 0 und Tag 7 dem zugesetzt.The protein-raised blood serum prepared according to the present invention was added to the explant of the subject explant group at
Am Tag 2, 7 und 14 wurde bei allen Testansätzen der Proteoglykangehalt im Kulturmedium gemessen. Hierzu wurde der Blyscan Gylcosaminoglycan Assay der Firma Biocolor Ltd., (Carrickfergus, UK) verwendet. Die Ergebnisse sind in der Graphik von
Die Analyse der Proteoglykanfreisetzung zeigt, dass in allen drei Gruppen, nämlich den 0-Kontrollen und den beiden stoßbehandelten/impaktierten Explantaten/ Knorpel-Knochen-Präparaten die Proteogykanmenge mit der Zeit ansteigt.The analysis of proteoglycan release shows that in all three groups, namely the 0 controls and the two shock-treated / impacted explant / cartilage-bone preparations, the amount of proteogycan increases with time.
Bei den mit dem erfindungsgemäß hergestellten proteinangereichten Blutserum behandelten Explantaten/Knorpel-Knochen-Präparaten ist dieser Anstieg jedoch nur wenig stärker als bei den nicht-impaktierten Kontrollen (0-Kontrollen), während die stoßbehandelten/impaktierten Explantate (Knorpel-Knochen-Präparate) ohne entsprechende Serumbehandlung einen sehr viel höheren Anstieg zeigen.However, in the explant / cartilage-bone preparations treated with the proteinaceous blood serum prepared according to the invention, this increase is only slightly greater than with the non-impacted controls (0 controls), while the shock-treated / impacted explants (cartilage-bone preparations) are without corresponding serum treatment show a much higher increase.
Dieser Test belegt somit den chondroprotektiven Effekt des erfindungsgemäßen Arzneimittels.
Claims (6)
- Cytokine- and gelsolin-enriched blood for use as a medicament, obtainable by collecting blood in a container that contains gold particles, and incubating said mixture of blood and gold particles.
- Blood for use according to claim 1, characterized in that after incubation the gold particles are removed from the blood and discarded.
- Blood for use according to claim 1, characterized in that gold particles and blood cells and/or other insoluble components are removed from the blood and discarded.
- Blood for use according to any one of claims 1 to 3 , characterized in that the gold particles have a size of about 1 µm.
- Blood for use according to any one of claims 1 to 4, characterized in that the gold particles are present in an amount of 103 - 104 gold particles per 10 ml container or in an amount of 0,3 mg gold particles with a diameter of 1 µm per 1 ml of blood/container.
- Blood according to any one of claims 1 to 5, for use in the treatment of osteoarthrosis.
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